Reconstitutable lyophilized protein formulation

ABSTRACT

A formulation for proteins is provided that comprises a lyophilized mixture of a protein, a water-soluble etherified cellulose in an amount that upon reconstitution of the formulation will be sufficient to form a gel, and an excipient to facilitate rehydration of the gel and to maintain protein integrity during storage of the lyophilized gel product. When reconstituted to a gel, this formulation can be applied, for example, to tissue in need of treatment.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to a lyophilized protein formulation, a gelreconstituted from the formulation with water, and a method of topicaltreatment utilizing the gel.

2. Description of Background and Related Art

In the past ten years, advances in biotechnology have made it possibleto produce a variety of proteins using recombinant DNA techniques forpharmaceutical applications. Because of proteolytic degradation in thegastrointestinal tract and poor permeability of these large molecularweight molecules through the intestinal mucosa, oral administration isusually not feasible. Hence, most of these proteinaceous pharmaceuticalagents are administered by intravenous, intramuscular, or subcutaneousinjections. The parenteral mode of delivery is also desirable for itsinherent pulsed administration. Banerjee, P. S., Parenteral Delivery ofPeptide and Protein Drugs, in Peptide and Protein Drug Delivery,Advances in Parenteral Sciences: 4. Lee V. H. L., ed. (Marcel Dekker,Inc., New York, N.Y., 1991)

Understandably, this form of administration has been poorly accepted bypatients, except for those suffering from life-threatening situations.For this reason, some proteins have recently been evaluated for topicalapplications, including relaxin for inhibition of uterine myometrialcontraction and cervical ripening at parturition, tissue factor forclotting blood, TGF-β for wound healing, and interferon-gamma for atopicdermatitis and trauma-related infections.

Methylcellulose is a synthetic derivative of the naturally occurringcellulose polymer. It differs from cellulose in that two of the threehydroxy groups of the glucose unit are substituted by a methoxy group.This group substitution allows cellulose to hydrate and form a hydrogel.In the pharmaceutical industry, this gel has been commonly used forsynthetic drugs and small peptides in topical applications. See, e.g.,Howell et al., J. Periodont. Res., 26: 180-183 (1991); Spirtos et al.,Gynecol. Oncol., 37: 34-38 (1990); Sacks et al., J. Infect. Dis., 161:692-698 (1990); Groeneboer et al., Curr. Eye Res., 8: 131-138 (1989);Bohr et al., Arch. Dermatol. Res., 279: 147-150 (1987); Schultz et al.,Cornea, 7: 96-101 (1988); Schmidt et al., Acta Ophthalmol., 59: 422-427(1981).

In addition, cellulose derivatives have been used to formulatetherapeutic proteins or polypeptides for topical use. See, e.g., EP267,015 published May 11, 1988; EP 308,238 published Mar. 22, 1989; andEP 312,208 published Apr. 19, 1989, which disclose formulation of apolypeptide growth factor having mitogenic activity, such as TGF-β, in apolysaccharide such as methylcellulose; EP 261,599 published Mar. 30,1988 disclosing human topical applications containing growth factorssuch as TGF-β; EP 193,917 published Sep. 10, 1986, which discloses aslow-release composition of a carbohydrate polymer such as a celluloseand a protein such as a growth factor; GB Pat. No. 2,160,528 grantedMar. 9, 1988, describing a formulation of a bioactive protein and apolysaccharide; and EP 193,372 published Sep. 3, 1986, disclosing anintranasally applicable powdery pharmaceutical composition containing anactive polypeptide, a quaternary ammonium compound, and a lower alkylether of cellulose. See also U.S. Pat. No. 4,609,640 issued Sep. 2, 1986disclosing a therapeutic agent and a water-soluble chelating agentselected from polysaccharides, celluloses, starches, dextroses,polypeptides, and synthetic polymers able to chelate Ca and Mg; and JP57/026625 published Feb. 12, 1982 disclosing a preparation of a proteinand water-soluble polymer such as soluble cellulose. In addition, amethod for entrapping enzymes in gel beads for use as a biocatalyst isdescribed in U.S. Pat. No. 3,859,169. Also, a method for preparingpolyvinyl alcohol gel intended as a transdermal vehicle forwater-soluble synthetic drugs is disclosed in JP 62/205035 publishedSep. 9, 1987.

TGF-β is typically formulated at an acidic pH at which it is active.Various methods for its formulation include adding 2-5% methylcelluloseto form a gel [Beck et al., Growth Factors, 3: 267-275 (1990) reportingthe effects on wound healing of TGF-β in 3% methylcellulose], addingcollagen to form an ointment or suspension [EP 105,014 published Apr. 4,1984; EP 243,179 published Oct. 28, 1987; EP 213,776 published Mar. 11,1987], or adding a cosmetically acceptable vehicle to the TGF-β for atopical formulation [U.S. Pat. No. 5,037,643 issued Aug. 6, 1991].

Additionally, proteins other than TGF-β have been formulated withcelluloses for various purposes. For example, epidermal growth factor(EGF) is mixed with a water-soluble cellulose polymer to obtain asterile aqueous medicinal composition. U.S. Pat. No. 4,717,717 issuedJan. 5, 1988. Also, certain pharmaceuticals are mixed with gelatin,lysozyme, albumin or skim milk along with a hydrophilic polymer such asmethylcellulose or hydroxypropyl cellulose to improve their absorptionor dissolution rate. JP 57/026615 published Feb. 12, 1982. Moreover,corticosteroids can be formulated with gelatin and methylcellulose orother celluloses to form a non-oil ointment. JP 61/233617 published Oct.17, 1986. In addition, cellulose derivatives have been used as a gelbase for relaxin. WO 89/07945 published Sep. 8, 1989. This publicationindicates that the formulated relaxin may be in liquid, frozen, or gelform, or may be lyophilized and reconstituted.

It has also been known to mix an active medicament unstable to heat witha biodegradable protein carrier such as collagen, atelocollagen, orgelatin to form a carrier matrix having sustained-release properties.The resultant mixture is then dried, and the dried material is formedinto an appropriate shape, as described in U.S. Pat. No. 4,774,091.Examples of active drugs for this purpose are given as t-PA;prostaglandins; prostacyclines; biohormones, e.g., hGH, bGH, GRF,somatomedins, and calcitonin; interferons; interleukins; tumor necrosisfactor; and other cytokines such as macrophage activating factor,migration inhibitory factor, and colony stimulating factor.

The major problem with incorporating proteins into gels is theinstability of these proteins in such a configuration. For example, whenrelaxin is incorporated into the liquid methylcellulose gel medium, theprotein is stable at 5° C. for only a month even in the absence oflight. In addition, for those proteins such as relaxin and TGF-β, theliquid containing the protein must be mixed with the gel at the time ofadministration. This is typically accomplished by the cumbersome andtime-consuming procedure of intermixing the two components throughsyringes connected by an interlocking unit. Insufficient mixing may alsolead to therapeutic failure.

For example, the current gel formulation package for relaxin containsone protein vial (lyophilized or liquid), one diluent vial (if theprotein is in lyophilized form), one liquid gel vial, two syringes, twoneedles, and one interlock connector for the syringes. In hospitals,physicians are required to undertake a complicated procedure of mixingthe protein and wet gel together, reconstituting the protein vial withdiluent, withdrawing reconstituted protein solution into one syringefirst, and then withdrawing wet gel into another syringe. After removingneedles from both syringes, they connect the syringes with an interlockconnector and push two syringe plungers back and forth to allow the geland protein solution to mix for use.

Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. Rey, "Some basic facts aboutfreeze drying," p. xiii, in Goldblith et al., ed., Freeze Drying andAdvanced Food Technology, Academic Press, London, 1975; Pikal,Freeze-drying of Proteins, parts 1 and 2, BioPharm, Sep. 1990, p. 18-27and Oct. 1990, p. 26-30. An excipient may be added to a formulation tobe freeze dried so as to reduce the time for reconstitution. Examples ofsuch agents include a sugar, polyol, amino acid, methylamine, orlyotrophic salt. See, e.g., Carpenter and Crowe, "The Mechanism ofCryoprotection of Proteins by Solutes," Cryobiology, 25: 244-255 (1988).

Various small molecular weight drugs have been formulated and thenfreeze-dried. A dried composition and method of oral administration ofdrugs, biologicals, nutrients, and foodstuffs is described in U.S. Pat.5,039,540; however, while the term freeze drying is used, the patentemploys a different process entirely. U.S. Pat. No. 4,883,785 disclosesa method for preparing a formulation for anti-fungal agent such asamphotericin B, where cyclodextrin is used to improve the solubility ofthe drug, and lyophilization is employed to preserve the formulation inthe solid state.

A freeze-drying method for preparing dry sponge-like polymeric carriersis described in JP 56/25211 published Jun. 24, 1980 by which embeddedenzymes (lysozyme, dextranase, mutanase, levanase) can be slowlyreleased when applied to the affected area of the mucous membrane insidethe mouth in treating oral cavity and naval cavity diseases.

Moreover, a method is disclosed for preparing lyophilized celluloselamellae containing low molecular weight molecules, such as pilocarpinechloride, for slow release in the ophthalmic field. Zaloukal,Ceskoslovenska Farmacie, 39: 433-435 (1990).

In contrast to small molecular weight drugs, proteins have a highmolecular weight associated with extensive secondary, tertiary, andquaternary structure that can be disrupted upon lyophilization. Hence,proteins tend to aggregate and/or deamidate when treated under certainconditions. There is a special need in the art for a topical,water-soluble polysaccharide-based gel formulation of protein that isstable, does not require complicated mixing procedures beforeapplication, and rehydrates readily from a powder reconstituted inwater.

Accordingly, it is an object of the present invention to provide atopical gel formulation for proteins that is stable for shipping andstorage and is easy to prepare prior to administration.

It is another object to provide a lyophilized formulation of proteinthat does not require a long time for reconstitution.

It is a further object to provide a formulation package having only oneproduct vial (containing lyophilized protein and gel base), one diluentvial, one syringe, and one needle. Such a package provides advantages inproduct packaging and manufacturing costs.

These and other objects will become apparent to one of ordinary skill inthe pharmaceutical, pharmacologic, veterinary, and clinical arts.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a formulation comprising alyophilized mixture of a protein, an excipient, and a water-solubleetherified cellulose in an amount that upon reconstitution of themixture will form a gel. This formulation is suitably reconstituted to agel with sufficient water.

In another embodiment, the invention provides a method for treatingtissue comprising the steps of:

(a) providing a formulation comprising a lyophilized mixture of aprotein, an excipient, and a water-soluble etherified cellulose in anamount that upon reconstitution of the mixture will form a gel,

(b) reconstituting the mixture in a sufficient amount of water to form agel, and

(c) applying a therapeutically effective amount of the gel topically tothe tissue.

In yet another embodiment, the invention provides a method of modulatingthe reproductive physiology of mammals during pregnancy and parturitioncomprising administering to the cervix or vagina a therapeuticallyeffective amount of the reconstituted gel formulation containing relaxinas the protein.

In a still further embodiment, the invention supplies a multi-unitformulation package comprising, in one unit, the lyophilized formulationbefore reconstitution, and in another unit, a diluent (reconstitutionagent) for the formulation. Additionally, the formulation may bemarketed as a dual-compartment syringe wherein one compartment containsthe lyophilized formulation before reconstitution and the othercompartment contains a diluent for the formulation.

Thus, the above formulation allows the use of only one product vial, onediluent vial, one syringe, and one needle. Gel preparation only involvesreconstitution of the lyophilized material with diluent and withdrawalof the reconstituted gel/protein mixture into a single syringe. Thiseliminates the need for an interlock connector for the syringes and forgel mixing in the syringes. The advantages in product packaging andfield application are obvious.

Lyophilized gel also is convenient for shipping and storage. Some wetgels tend to lose viscosity under light and heat stress, due possibly toa trace impurity in a wet gel that can initiate degradation of gelpolymers, which reaction quickly propagates in a liquid environment.Since lyophilized gels are in a solid form, the opportunity for traceimpurities to propagate this reaction is minimized.

In addition, lyophilized gel can reduce the manufacturing costsignificantly. Lyophilized gels require only one filling operation inwhich the mixture of protein and gel fluid is filled into a vial forlyophilization. One filling line operation requires much less equipmentcapital and operating cost than two filling lines required for proteinsolution in one vial and wet gel in the other.

Furthermore, reconstitution time can be as short as 10 minutes due tothe presence of the excipient. Additionally, the effect oflyophilization on the protein is not detrimental to the protein when theformulation is reconstituted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a graph of log resistivity versus temperature and howglass transition temperature is determined from resistivity for a 3%(w/v) hydroxyethyl cellulose gel so as to obtain primary dryingtemperature for gel lyophilization.

FIG. 2 illustrates a graph of ultraviolet absorbance versus time for a1% (w/v) hydroxyethyl cellulose gel containing 5 mM polyethylene glycol(PEG) 1000 so as to determine reconstitution time for lyophilized gel.

FIG. 3 illustrates a graph of viscosity versus shear rate to show theeffect of the autoclave on the viscosity of 3% and 4% (w/v) hydroxyethylcellulose (HEC) gels.

FIG. 4 illustrates a graph of viscosity versus shear rate to determinethe concentration of hydroxyethyl cellulose (HEC) gel that would givethe same viscosity as a 3% (w/v) methylcellulose (MC) gel.

FIG. 5 depicts the reconstitution times of 1% (w/v) hydroxyethylcellulose gels containing differing concentrations of glycerol ordifferent grades of PEG as excipients as determined by uv absorbance.The control is the hydroxyethyl cellulose gel alone or the gel withmethionine.

FIG. 6 depicts the reconstitution times of 3.5% (w/v) hydroxyethylcellulose gels containing four different excipients in a range ofconcentrations.

FIG. 7 depicts the stability of relaxin in 3.5% (w/v) hydroxyethylcellulose gels containing glycerol or PEG 300 at 5° C. or 30° C. asmeasured by reverse-phase HPLC.

FIG. 8 represents a graph of the stability of relaxin in 3.5% (w/v)hydroxyethyl cellulose gels containing different excipients at 5° or 25°C. as measured by reverse-phase HPLC.

FIG. 9 represents a graph of the stability of relaxin in 3.5% (w/v)hydroxyethyl cellulose gels containing different excipients at 5° or 25°C. as measured by ELISA.

FIG. 10 represents a graph of the stability of tissue factor in 3.5%(w/v) hydroxyethyl cellulose gels containing glycerol or differentgrades of PEG at 5° C. as measured by size-exclusion HPLC.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The basic components of the gel formulation of this invention are aprotein, a water-soluble etherified cellulose, and an excipient. Each ofthese components is addressed more fully below.

By "protein" is meant a sequence of amino acids for which the chainlength is sufficient to product the higher levels of tertiary and/orquaternary structure. This is to distinguish from "peptides" or othersmall molecular weight drugs that do not have such structure. Typically,the protein herein will have a molecular weight of at least about 15-20kD, preferably at least about 20 kD.

Examples of proteins encompassed within the definition herein includemammalian proteins, such as, e.g., a growth hormone, including humangrowth hormone, des-N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroid stimulating hormone; thyroxine;lipoproteins; α1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; leutinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial naturietic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumornecrosis factor-alpha and -beta; enkephalinase; a serum albumin such ashuman serum albumin; mullerian-inhibiting substance; relaxin A-chain;relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; amicrobial protein, such as beta-lactamase; DNase; inhibin; activin;vascular endothelial growth factor; receptors for hormones or growthfactors; integrin; thrombopoietin; protein A or D; rheumatoid factors; aneurotrophic factor such as bone-derived neurotrophic factor (BDNF),neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nervegrowth factor such as NGF-β; platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-γ5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); insulin-like growth factor bindingproteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; a bone morphogenetic protein(BMP); somatotropins; an interferon such as interferon-alpha, -beta, and-gamma (IFN-α, -β, and -γ); colony stimulating factors (CSFs), e.g.,M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; antibodies; and fragments of any of theabove-listed polypeptides. Preferred proteins herein are therapeuticproteins when applied topically, particularly those having a therapeuticeffect when applied topically to treat tissue. These include growthfactors such as TGF-β, TGF-α, PDGF, EGF, FGF, and IGF-I, plasminogenactivators such as t-PA, clotting factors such as tissue factor andfactor VIII, hormones such as relaxin and insulin, or cytokines such asIFN-γ, most preferably tissue factor, relaxin, and TGF-γ.

"IFN-γ" is meant to include IFN-γ in a mature, pro, met, or des(1-3)(also referred to as desCysTyrCys IFN-γ) form, whether obtained fromnatural sources, chemically synthesized, or produced by techniques ofrecombinant DNA technology. A complete description of the preparation ofrecombinant human IFN-γ, including its cDNA and amino acid sequences, isprovided in U.S. Pat. Nos. 4,762,791; 4,929,544; 4,727,138; and4,925,793. CysTyrCys-lacking recombinant human IFN-γ interferons,including variously truncated derivatives, are disclosed, for example,in EP 146,354. Non-human animal IFN-γ is, for example, disclosed in EP88,622. The term includes variously glycosylated forms and othervariants and derivatives of such interferons, whether they are known inthe art or will become available in the future. Examples of suchvariants include alleles and the products of site-directed mutagenesisin which residues are deleted, inserted, and/or substituted (see, e.g.,EP 146,354).

By "TGF-β" is meant the family of molecules that have either thefull-length, native amino acid sequence of any of the TGF-βs from anyspecies, including the latent forms and associated or unassociatedcomplex of precursor and mature TGF-β ("latent TGF-β"). Reference tosuch TGF-β herein will be understood to be a reference to any one of thecurrently identified forms, including TGF-β1, TGF-β2, TGF-β3, TGF-β4,and TGF-β5 and latent versions thereof, as well as to TGF-β speciesidentified in the future, including members of the TGF-β family. Membersof the TGF-β family are defined for purposes herein as those which havenine cysteine residues in the mature portion of the molecule, share atleast 70% homology, preferably at least 80% homology, with other knownTGF-β sequences in the mature region, and compete for the same receptor.In addition, they all appear to be encoded as a larger precursor thatshares a region of high homology near the N-terminus and showsconservation of three cysteine residues in the portion of the precursorthat will later be removed by processing. Moreover, the TGF-βs appear tohave a four or five amino acid processing site. The TGF-β isappropriately from any source, preferably mammalian, and most preferablyhuman for treating humans. TGF-β from animals other than humans, forexample, porcine or bovine sources, can be used for treating humans.Likewise, if it is desirable to treat other mammalian species such asdomestic, farm, zoo, sports, or pet animals, human TGF-β, as well asTGF-β from other species, is suitably employed.

"Relaxin" denotes a functional protein having specific hormonalfunctions, such as, for example, the modulation of the reproductivephysiology of human beings and possibly other mammals, including, butnot limited to, maintaining pregnancy, effecting parturition, andenhancing sperm motility as an aid in fertilization. Human relaxin andits methods of preparation, including synthesis in recombinant cellculture, are known. EP 101,309 and 112,149. In addition, relaxin may beprepared by synthesis of the A and B chains, and purification andassembly thereof, as described in EP 251,615 published Jan. 7, 1988. Theterm includes relaxins from native or recombinant sources as well asrelaxin variants, e.g., those with insertions, substitutions, ordeletions of one or more amino acid residues, glycosylation variants,the prepro- and pro- forms, etc. These variants include those having 33amino acids in the B chain rather than the 29 found in the naturalmolecule. The preferred relaxin herein is human relaxin.

"Tissue factor" refers to a protein capable of correcting variousbleeding disorders, e.g., by inducing coagulation, particularly thosedisorders associated with coagulation factor deficiencies. Includedwithin this term is human tissue factor protein having nativeglycosylation and the amino acid sequence set forth in FIG. 2 of EP278,776 published Aug. 17, 1988, analogous tissue factor proteins fromother animal species such as bovine, porcine, ovine, and the like,deglycosylated or unglycosylated derivatives of such tissue factorproteins, and biologically active amino acid sequence variants of tissuefactor, including alleles and in vitro-generated covalent derivatives oftissue factor proteins that demonstrate at least one of its activities.The preferred tissue factor herein is human tissue factor.

The "water-soluble etherified cellulose" herein is a cellulose etherpolysaccharide that constitutes the gelling agent for the proteinformulation herein. The term "water soluble" as applied to theetherified cellulose herein is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient.

Examples of etherified cellulose herein include alkyl celluloses,hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses, for example,methylcellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose,and hydroxypropyl cellulose. The preferred gelling agent herein is onethat is inert to biological systems, non-ionic, non-toxic, simple toprepare, and not very runny or viscous, will not destabilize the proteinheld within it, and allows good rehydration of lyophilized gel. It isnoted that the preferred cellulose derivatives herein are those that arenot anionic, such as those in the form of alkali metal salts, e.g., Li,Na, K, or Cs salts, or carboxymethyl cellulose, to avoid anyinactivation of the protein, particularly for TGF-β.

Preferably the etherified cellulose is well defined, purified, andlisted in USP, e.g., methylcellulose and the hydroxyalkyl cellulosederivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose,and hydroxypropyl methylcellulose. Most preferred are those cellulosederivatives with highly polar substituted side chains, which show betterrehydration properties than non-polar forms such as methylcellulose. Themost preferred cellulose herein is hydroxyethyl cellulose due to itsshorter reconstitution time as well as its ease of preparation andsterilization by autoclaving.

An "excipient" is an agent whose principal function is the accelerationof reconstitution of the lyophilized mixture (i.e., it reduces gelrehydration time). A secondary function of the excipient is its abilityto stabilize the protein, i.e., it maintains or improves proteinstability. In a polymer solution, the flexible chains of dissolvedmacromolecules interpenetrate and entangle because of the constantBrownian motion of their segments. In an aqueous solution, thefunctional groups of each chain are encased in a solvating sheath ofhydrogen-bonded water molecules. This envelope of water of hydrationprevents neighboring chain segments from forming interchain hydrogenbonds.

Without being limited to any one theory, it is believed that when thiswater is removed during lyophilization, the polymer molecules are nowfree to associate and form hydrogen and/or hydrophobic bonds that may beirreversible, or at the very least, only slowly reversible uponsubsequent rehydration. This would explain why lyophilizedmethylcellulose gels take up to two days to rehydrate. Certainexcipients preferentially hydrate other macromolecules in aqueoussolutions and in the solid state as well. In addition, lyophilization ofmethylcellulose gels containing such excipients requires lower operatingshelf temperatures as well as extended drying times. The resulting cakesalso exhibit lower collapse temperatures and higher final moisturecontents. These are all indications of stronger interaction betweenwater and cellulose molecules. The rehydration of these cakes takesabout two hours versus two days for samples not containing theexcipient. By maintaining the hydration sheath throughout the dryingprocess, most of the interchain bonding seems to be eliminated, so thatthe gel can rehydrate fully and quickly.

The particular excipient used for this purpose will depend on thespecific protein being formulated, but preferably is a sugar such assucrose, an amino acid such as monosodium glutamate, a methylamine suchas betaine, a lyotrophic salt such as magnesium sulfate, or a polyolsuch as trihydric or higher sugar alcohols, most preferably glycerin,erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol, as wellas propylene glycol. See, e.g., Carpenter and Crowe, supra. Thepreferred excipient herein is one that reduces the rehydration time ofthe lyophilized mixture to less than 30 minutes (i.e., is clinicallyviable) without adversely affecting the stability of the protein. Forrelaxin and TGF-β, the excipient of choice is sucrose, whereas fortissue factor, the preferred excipient is glycerol.

Additional additives may be added to the formulation as desired forimparting certain other properties, including a co-solvent,anti-oxidant, or agent to enhance absorption of the protein by thetissue. For example, relaxin may be formulated with an anti-oxidant suchas methionine (e.g., in amounts of about 0.01 to 0.1% w/v of the gel) orascorbic acid (about 0.3-1% w/v of the gel), or with a co-solvent suchas glycerol (about 0.1-20% w/v of the gel), and/or ethanol (about0.1-20% w/v of the gel) to minimize oxidation of the protein by light.These agents can be used alone or in a combination thereof, preferablyin an amount of about 1 to 25% by weight, preferably 2 to 10% by weight,of the gel, taking into account the amounts of the other ingredients.The preferred of these agents for relaxin is glycerol or ethanol, in anamount of 0.1 to 10% (w/v) of the gel.

Examples of agents to enhance absorption (as by the cervix or vagina ifthe protein is relaxin) include molecules with a structure similar tothat of cholesterol, such as glycocholate, e.g., sodium glycocholate,cholate, e.g., sodium cholate, and fusidic acid and its derivatives,including salts and esters such as 24,25-tourodihydrofusidate, non-ionicsurfactants, derivatives of fatty acids having about 7 to 25 carbonatoms, such as oleic acid, niacinamide, nicotinic or salicyclic acid, ortheir salts or esters, and azone.

The formulation herein also may suitably contain more than one proteinas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect theother protein. For example, those proteins with mitogenic or angiogenicactivity include, e.g., TGF-β, IGF-I or -II, TGF-α, human growthhormone, EGF, vascular endothelial growth factor, vaccinia virus growthfactor, acidic or basic FGF, angiogenin, NGF, PDGF, human endothelialcell growth factor, or fibronectin. Such proteins are suitably presentin combination in amounts that are effective for the purpose intended.

The respective amounts of the various required components in theformulation, and the preferred amounts, are indicated below.

The protein formulations herein will be dosed in a fashion consistentwith good medical practice taking into account the nature of the tissueto be treated, the type of protein, the indication to be treated, thespecies of the host, the medical condition of the individual patient,the presence of any other co-treatment drug in the composition, the siteof delivery of the protein, the scheduling of administration, and otherfactors known to practitioners. Because of differences in host response,significant site-to-site and patient-to-patient variability exists. Forpurposes herein, the "therapeutically effective amount" of protein orgel is an amount that is effective to treat soft or hard tissue for theintended purpose of the protein at the desired site.

As a general proposition, depending on the protein type, the protein isformulated at an amount of about 0.010 μg/ml-5 mg/ml, preferably about0.2 to 1 mg/ml, of the gel (wet gel, before lyophilization). If theprotein is TGF-β, preferably, the amount is about 0.025 μg per ml to 1mg per ml of gel, most preferably about 0.2-1 mg per ml of gel. If theprotein is relaxin, the preferred amount is about 0.1 to 1 mg/ml of gel.If the protein is tissue factor, the preferred amount is about 0.2 to 1mg/ml of gel, and the preferred dose is about 1-25 μg/kg. If the proteinis IFN-β, the preferred dose is about 0.01 mg/M² to 0.1 mg/M² per dayfor atopic dermatitis.

As noted above, these suggested amounts of protein are subject to agreat deal of therapeutic discretion. The key factor in selecting anappropriate dose is the result obtained, for which veterinary orclinical parameters are used to determine an indication-appropriateendpoint. For example, the formulation herein is useful in the treatmentof soft or hard tissue, generally in mammals and preferably humans,preferably not ocular tissue, and preferably skin or bone. For TGF-βthis would include the promotion of surface wound healing, and thetopical application to internal surgical incisions. Examples of suchapplications include treatment of burns; surface ulcers, includingdecubital, diabetic, dental, hemophiliac, and varicose ulcers; wounds,including lacerations, incisions, and penetrations; surgical incisions,including those of cosmetic surgery; and bony defects or repair. Forrelaxin, these indications include cervical ripening at parturition andinhibition of uterine myometrial contraction. For t-PA, this treatmentwould include healing of adhesions. For IFN-β, these indications includeatopic dermatitis, other rashes, and trauma-related infections that canbe treated topically. For tissue factor, this treatment includeshemostat application to stop bleeding. The measurements of veterinaryand clinical parameters for these indications are well known to thoseveterinarians, clinicians, and pharmacologists skilled in the art.

The cellulose derivative must be present in the formulation insufficient quantities that a gel of the proper viscosity will be formedupon reconstitution for topical application. The amount of etherifiedcellulose employed will depend mainly on the molecular weight and theviscosity of the particular etherified cellulose being employed. Ingeneral, the higher the viscosity and molecular weight of the cellulosederivative, the lower the amount needed in the gel. The lower amount ofsuch derivative is generally about 1% (w/v) of the gel (beforelyophilization of the mixture) for higher viscosity derivatives, and theupper limit of such amount is determined by the maximum amount oflow-viscosity or low-molecular-weight etherified cellulose that can beadded to the formulation that will still maintain the effectiveness ofthe gel. The cellulose derivative is preferably present in the gelformulation in the range of about 1-20% (w/v) of the gel, depending onthe viscosity and molecular weight, more preferably about 1-15%, andmost preferably about 2-10% of the gel. If the etherified cellulose is ahigh-viscosity derivative (e.g., methylcellulose of the A4M Type fromDow Chemical or hydroxyethyl cellulose under the tradename Natrosol),then the preferred range is about 2-5% (w/v) of the gel, and mostpreferably about 3-4% (w/v).

The concentration of excipient will depend mainly on the type ofexcipient and protein employed. If sugar is the excipient, it ispreferably present in amounts of about 0.1 to 2M of the gel (beforelyophilization of the mixture), and if glycerol is the excipient, it ispreferably present in amounts of about 0.5 to 5% (w/v) of the gel, morepreferably about 1-4% (w/v). The determination of the amount to employis made by examining cake appearance, stability, and reconstitutiontime.

The particularly preferred formulations herein, based on volume of totalgel before lyophilization, are presented below. The preferred relaxinformulation is about 0.2-0.5 mg/ml, preferably about 0.2 mg/ml, relaxin,about 3-4% (w/v), preferably about 3.5% (w/v), hydroxyethyl cellulose,and about 0.2-0.6M, preferably about 0.2M, sucrose, optionally withabout 0.03-0.1% (w/v), preferably about 0.05% (w/v), free methionine.The preferred TGF-β formulation herein is about 0.2-1 mg/ml, preferablyabout 0.5% mg/ml, TGF-β, about 3-4% (w/v), preferably about 3.5% (w/v),hydroxyethyl cellulose, and about 0.2-0.6M, preferably about 0.2M,sucrose. The preferred tissue factor formulation herein is about 0.5-1mg/ml, preferably about 1 mg/ml, tissue factor, about 3-4% (w/v),preferably about 3.5% (w/v), hydroxyethyl cellulose, and about 1-4%(w/v), preferably about 1% (w/v), glycerol.

The formulation herein is preferably sterile. Sterility of the proteinis readily accomplished by sterile filtration of the protein in thebuffer through (e.g., 0.2 micron) membranes prior to mixing with theother ingredients. The protein ordinarily will be stored as an aqueoussolution prior to formulation and lyophilization, although lyophilizedformulations of the protein for reconstitution prior to admixture withthe etherified cellulose are also acceptable.

Sterility of the entire mixture is accomplished in one procedure byautoclaving the ingredients except for protein at about 120° C. forabout 30 minutes and storing the resulting mixture at 5° C. for about 24hours to allow the etherified cellulose to hydrate before mixing withthe sterile-filtered protein solution. Then the two mixtures arecombined and lyophilized for final formulation.

The active protein ingredient is generally combined at ambienttemperature at the appropriate pH, and at the desired degree of purity,with the other ingredients. The pH of the formulation will depend mainlyon the type of protein being formulated. In certain instances, as withTGF-β, relaxin, and IGF-I, the protein is generally formulated at anacidic pH, i.e., less than pH 7, preferably about pH 3-6.5, and morepreferably about pH 4-6.

Further, if the protein to be administered is TGF-β, to be effective, itis converted by the body to its activated form, i.e., the mature form iscleaved from its precursor using a suitable enzyme and the resultantcomplex is treated with acid or other appropriate agent, to activate theTGF-β. Nevertheless, TGF-β is suitably administered in an inactive ordelayed release form such as a complex of mature TGF-β with proTGF-β notcontaining mature TGF-β (i.e., the remaining precursor of TGF-β), with aTGF-β binding protein, or with alpha₂ -macroglobulin. The latent form isthen converted to the active form either by naturally occurringmechanisms in the local environment or by formulation with TGF-βactivating agents described above. See, e.g., Gentry et al., Mol. Cell.Biol., 4162-4168 (1988); Miyazono et al., J. Biol. Chem., 263: 6407-6415(1988); Wakefield et al., J. Biol. Chem., 263: 7646-7654 (1988);Keski-Oja et al., J. Cell Biochem. Suppl., 11A: 60 (1987);Kryceve-Martinerie et al., Int. J. Cancer, 35:553-558 (1985); Lawrenceet al., Biochem. Biophys. Res. Commun., 133: 1026-10134 (1985); Lawrenceet al., J. Cell Physiol., 121: 184-188 (1984). Thus, the pH of the TGF-βcomposition may suitably reflect the conditions necessary foractivation.

After the protein, etherified cellulose, and excipient are mixedtogether, the formulation is lyophilized. This may be accomplished byany suitable means, including ramping the temperature of the sample downto a temperature suitable for primary drying. This temperature isdetermined by the glass transition temperature of the formulatedcellulose gel without protein using resistivity measurements. Typically,the primary drying temperature will range from about -30° to -60° C. ata suitable pressure, ranging typically from about 130 to 170 μm,preferably about 150 μm. The size and type of the container holding thesample (e.g., cuvette or vial) will mainly dictate the time required fordrying, which generally ranges from about 1 to 7 days.

A secondary drying stage is then typically carried out at about 5°-20°C., depending primarily on the type and size of container 10 and thetype of protein employed. The time and pressure required for secondarydrying will be that which produces a suitable lyophilized cake,dependent, e.g., on the temperature and other parameters. Typically, thetime will take at least about 4 hours and the pressure will be the sameas that employed during the primary drying step.

At the desired stage, typically in a clinical setting when it is desiredthat the gel be applied to the patient, the lyophilized powder isreconstituted with sufficent diluent to rehydrate the gel for topicalapplication. Reconstitution generally takes place at a temperature ofabout 5°-10° C. to ensure complete hydration, although othertemperatures may be employed as desired. The time required forreconstitution will depend, e.g., on the type of excipient and protein.The diluent employed for this purpose is typically an aqueous mediumsuch as water, water for injection, Ringer's solution, or buffer.

The diluent is suitably contained in a separate vial in a pre-packagedkit that also contains a vial of the lyophilized formulation.Alternatively, the lyophilized mixture is suitably packaged in onecompartment of a dual-chamber syringe, and the other compartment isfilled with the diluent. An example of a dual-compartment syringesuitable for this purpose is that marketed by Preject Inc., Warwick,R.I.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

EXAMPLES

The protocols and methods described below were employed in the examplesthat follow:

A. Gel Preparation 1. Hydroxyethylcellulose (Natrosol)

The appropriate amount of water or a desired buffer was added to ameasured amount of Natrosol powder, either Natrosol 250 MR or Natrosol250 HR. The solution was then stirred at room temperature for at leastfour hours at medium speed until the solution gelled. The concentrationof the gel was 1% (w/v) and 3.5% (w/v) for reconstitution and stabilitystudies, respectively.

2. Methylcellulose or Hydroxypropyl Methylcellulose (Metolose)

The buffer was heated in a tissue culture flask while stirring. The heatwas turned off once the temperature had reached 85° C. Metolose USPpowder (Metolose SM-4000 or Metolose 90SH-4000) was then dispersed intothe hot solution. The flask was capped with a double-headed filter unitand autoclaved using the liquid cycle. After autoclaving, thedouble-headed filter unit was taken off in the laminar flow hood and aregular cap was put on. The gel was then stirred at 5° C. overnight andwrapped in foil the next day.

3. Hydroxypropylcellulose (Klucel HF)

An amount of water at least six times the dry weight of Klucel powderwas heated to 55° C. The dry Klucel powder was added to the hot waterwhile stirring. Continuous stirring was maintained for 15 minutes toensure uniform dispersion. Enough cold water was then added to make thedesired concentration. Stirring was continued at a slow speed untilgelation occurred.

B. Viscosity

Gel viscosity was measured with a Brookfield viscometer. Half amilliliter of the gel was placed in the sample cup with spindle CP-51.After equilibrating at 25° C. at the lowest rpm for 5 minutes, torquemeasurements of spindle were collected and calculated to obtainviscosity. The viscometer rotation was changed every 15 seconds in astepwise manner from 0.5 rpm to 100 rpm and then back down to 0.5 rpm.

C. Autoclave

Approximately 10 ml of each of the gels we re autoclaved in 50 mlbeakers at 121° C. for 30 minutes using the liquid cycle in the GetingePACS 50 autoclave.

D. Eutectic Temperature Determination

The primary drying temperature was based on the glass transitiontemperature of the formulated gel without protein as determined byresistivity measurements. An Ultra-Vu disposable cuvette was filled withjust enough gel to cover the resistivity probe, and the whole assemblywas placed in the Planer Biomed incubator. After equilibration at 5° C.for 10 minutes, the sample was cooled at 5° C. per minute to -60° C. Thesample was again equilibrated at this temperature for 15 minutes beforeit was warmed at 2° per minute to 20° C. The glass transitiontemperature was determined as shown in FIG. 1. This temperature is about-30° C. for a 3% (w/v) Natrosol 250 MR gel prepared in 10 mM citratewithout salt at pH 5.0.

E. Lyophilization

The general scheme for the lyophilization cycle was as follows:

Cool the lyophilizer to 5° C. in 15 minutes.

Keep the samples at 5° C. for 30 minutes.

Freeze in 2 hours to -55° C.

Hold samples at -55° C. for a minimum of two hours.

Ramp up to -30° C. in 2 hours for primary drying.

Conduct primary drying at -30° C. at a pressure of 150 μm.

The betaine samples were frozen in a -70° C. freezer overnight andtransferred to the lyophilizer only after the shelves had reached -55°C. This procedure was necessary to prevent collapse of these samplesduring primary drying.

The duration of primary drying depended on whether cuvettes or vialswere used. If cuvettes were used, primary drying required up to 5 to 6days, whereas if vials were used, less than two days were needed.

Conduct secondary drying at either 20° C. (cuvettes) or 5° C. (vials)for at least 4 hours at the same pressure. Vials were heated only to 5°C. so that they would be at the same temperature as the controls used inthe stability studies.

If the vials contained active materials, then upon conclusion of thelyophilization run, the vials were subjected to aultra-high-purity-grade nitrogen gas atmosphere before sealing.

F. Cuvette Fill

The gels containing different excipients were filled from a syringe withan 18-gauge needle into the disposable cuvettes until they were justabove the narrowest part of the cuvette (approximately 1 g of gel). Fivecuvettes of each excipient were filled. The weights of the empty andfilled cuvettes were recorded. The cuvettes were then arranged intosmall groups and placed on the middle shelf of the lyophilizer.

G. Reconstitution of Cuvettes

The lyophilized gel in its cuvette was weighed and the difference fromthe weight of the cuvette filled with non-lyophilized gel was noted. Itwas assumed that the loss in weight was solely due to sublimation. Thisloss was then replaced with the same volume of water to reconstitute thelyophilized gel to its original concentration. The water was addedslowly along the side of the cuvette to minimize bubble formation.

H. Reconstitution Time Determination

The uv absorbance of the reconstituted gel at 340 nm was measured overtime using the Hewlett-Packard spectrophotometer. The spectrophotometerwas zeroed against water as a reference solvent. Absorbance was measuredimmediately after water was added to the lyophilized gel andmeasurements were taken thereafter at one-minute intervals. A two-hourtime frame was usually allotted to ensure complete reconstitution of thelyophilized gel. Reconstitution was judged to be complete when theabsorbance readings leveled off. The reconstitution time was thendetermined as shown in FIG. 2.

I. Protein Preparation

Depending on the status of the protein (i.e., concentration and buffersystem), preparation may or may not be necessary. If the proteinconcentration was too low, then the bulk solution was concentrated withan Amicon Stirred Cell to more than 10 times the required concentrationfor HPLC assays with a 10K molecular weight cut-off filter. Proteinconcentration was measured with the spectrophotometer over the range of240 to 400 nm. If the buffer system was not appropriate, then the bulksolution was dialyzed against the desired buffer system at 5° C. forabout 24 hours.

J. Gel Preparation and Vial Fill

Excipients, hydroxyethyl cellulose powder, and protein were combinedwith water to make a 3.5% (w/v) cellulose solution. The solutions werethen stirred at room temperature for at least 4 hours at medium speeduntil the solutions gelled. The gels were filled with a 181/2 gaugesyringe into 3-cc vials. Approximately 0.5 g of the gels were filledinto each vial. The weights of the empty and filled vials were recorded.

K. Stability Schedule

Two vials per time point were set up for each stability study. Timepoints were at one-week intervals. After Time 0 the vials were dividedinto two groups that were stored at 5° C. and 30° C., respectively.

L. Reconstitution and Dilution for HPLC Study

The same reconstitution procedure as the one for cuvette reconstitutionwas followed. The gels were reconstituted at 5° C. overnight to ensurecomplete rehydration. The reconstituted gels were then diluted 10 timesthe next day with the appropriate buffer for the intended HPLC assay.The diluted gels were vortexed at a setting of 10 on the Vortex Mixerfor at least 2 minutes to ensure that the gel was completely dissolved.

EXAMPLE I

This example illustrates the invention using relaxin as the protein.

A. Reverse-Phase HPLC Assay for Relaxin

Instrument: HP 1090

Column: Vydac C4

Mobile Phase: A) 0.1% trifluoroacetic acid (TFA) in water, B) 0.1%TFA/90% acetonitrile

Gradient: 20% to 50% B in 30 minutes

Flow Rate: 1.0 ml/min

Injection Volume: 100 μl

Protein Concentration: 20 μg/ml

UV Detector: 214 nm

Run Time: 37 min.

Post Time: 15 min.

Temperature: Ambient

B. Relaxin ELISA

This assay determines the concentration of intact relaxin. The liquidrelaxin and the lyophilized gel were diluted with relaxin diluent(phosphate buffered saline, 0.5% bovine serum albumin, 0.05%polysorbate, 0.01% thimerosal) to 5 ng/ml, 2.5 ng/ml, and 1.25 ng/ml.Duplicate aliquots of the diluted samples were submitted for analysisusing antibodies to relaxin.

C. Study No. 1: Effects of Different Grades of PEG and Glycerol onReconstitution Time of the Gel

Previous studies have shown that polyethylene glycol (PEG) 300 shortensreconstitution time. This study further explores this phenomenon usingdifferent grades and concentrations of PEG as well as glycerol. Themajor route of relaxin degradation in methylcellulose gel has been foundto be through oxidation of methionine. To reduce this possibility, 0.05%(w/v) free methionine was included in all the relaxin formulationsprepared hereafter. The combination of methionine and glycerol was foundto be protective against relaxin degradation, so these components werefirst tested in the 1% w/v hydroxyethyl cellulose gel. A 1% w/v gel wasmade because gels with higher concentrations often had trapped bubblesthat would interfere with absorbance measurements. The excipientsincluded in this study were:

1) glycerol: 0.1% w/v, 1% w/v, 5% w/v, 10% w/v

2) PEG 300: 5 mM, 10 mM, 25 mM,

3) PEG 600: 5 mM, 10 mM, 25 mM

4) PEG 1000: 5 mM, 10 mM, 25 mM

PEG 3350: 5 mM, 10 mM, 25 mM

D. Study No. 2: Effects of Different Classes of Excipients onReconstitution Time of the Gel

This study was initiated to test the different classes of excipientsthat have been shown to be stabilizers for proteins. The classes ofexcipients and their representatives tested in this study were:

1) Sugars - sucrose

2) Polyols - mannitol

3) amino acids - monosodium glutamate (MSG)

4) methylamines - betaine

5) Lyotrophic salts - magnesium sulfate

The concentrations prepared for each excipient were 0.2M, 0.4 M, 0.6M,0.8M, 1.0M, 1.5M, and 2.0 M. These concentrations were chosen becauseCarpenter and Crowe, supra, found that for these excipients the optimalsolute concentration for protein protection was between 0.2 and 1.8M.

E. Study No. 3: Effects of PEG. Glycerol, and Sucrose on Gel ContainingRelaxin

Synthetic human (H2) relaxin [Johnston et al., in Peptides: Structureand Function, Deber, C. M., et al. (eds.), p. 683-686, Pierce ChemicalCompany, Rockford, Ill., 1985] was employed. This product is currentlyavailable from Genentech, Inc., South San Francisco, Calif., to clinicalinvestigators for clinical trials. The excipients selected were 1% w/vglycerol and 50 mM PEG 300. The concentration of 1% w/v of glycerol wasfound in earlier studies to give a good lyophilized cake, and theconcentration of 50 mM PEG 300 was chosen because it was expected togive a reasonable reconstitution time. The sucrose concentration usedwas 0.2M. All gels contained 0.2 mg/ml of protein. Primary drying wascarried out at -40° C. for 56 hours and 30 minutes. Again, a three-weekstability study was set up. Reverse-phase HPLC of reconstituted gel wasused to monitor protein stability.

F. Results 1. Gel Selection

Of the five gels examined (Natrosol 250 MR, Natrosol HR, MetoloseSM4000,Metolose 90SH4000, and Klucel), only the two grades of Natrosolgels survived autoclaving. Viscosity was measured to confirm that therewas no degradation after autoclaving (FIG. 3). The MR grade was chosenbecause it can be prepared at the concentration that would provide thedesired viscosity.

2. Viscosity

Since the present study was based on the relaxin gel project, manyparameters were kept constant to reduce undesired variabilities in theexperiment. Viscosity of the gel was kept constant by adjustingcellulose concentration. From viscosity measurements the Natrosol gelconcentration corresponding to the 3% (w/v) methylcellulose gel wasfound to be 3.5% (w/v) (FIG. 4).

3. Study No. 1

Both the glycerol- and PEG-containing cakes were yellow afterlyophilization. All samples gave good cakes except for the gelscontaining 5% (w/v) glycerol or greater. However, the higherconcentrations of glycerol required the shortest reconstitution time. Asfor PEG, the higher the molecular weight and/or higher theconcentration, the shorter the reconstitution time (FIG. 5). The resultssuggest that a glycerol concentration greater than or equal to 1% w/vreduced the reconstitution time significantly.

4. Study No. 2

It was found from resistivity measurements that a solution of 2.0Mbetaine would not crystallize until it had reached a temperature of -70°C. Therefore, the betaine samples were frozen in a -80° C. freezerbefore transferring to the lyophilizer. As for the other excipients athigher concentrations, those listed below were insoluble, so they werenot prepared for lyophilization:

1) sucrose: 2.0M

2) mannitol: 1.0M, 1.5M, 2.0M

3) sodium glutamate: 1.5M, 2.0M

4) magnesium sulfate: 1.0M, 1.5M, 2.0M

Only sucrose and mannitol at low concentrations (<0.8M) gave goodlyophilized cakes. The others either melted back or were not"cake-like." As for those that were reconstituted, all but mannitol hada reconstitution time of less than 20 minutes (FIG. 6). Thereconstitution time of sucrose did not vary within the range ofconcentrations examined, whereas that of sodium glutamate decreased withincreasing concentration. However, mannitol had the opposite effect.Overall, this study demonstrates that sucrose is an appropriateexcipient to reduce gel reconstitution time to approximately 10 minutes.

5. Study No. 3

The results from this study indicate that neither PEG nor glycerolprotected the relaxin protein from degrading compared to the control atall time points (FIG. 7). However, sucrose shows the ability to protectrelaxin (FIG. 8). The ELISA assay confirms this result (FIG. 9).

EXAMPLE 2

This example illustrates the invention utilizing tissue factor as theprotein.

A. Size-Exclusion HPLC Assay for Tissue Factor

Instrument: HP 1090

Column: pharmacia Superose 12

Flow Rate: 0.5 ml/min

Mobile Phase: 0.8% (w/v) octylglucoside, 0.01MNaPO₄, 0.15M NaCl, pH 7.3

Injection Volume: 50 μl

Protein Concentration: 0.1 mg/ml

UV Detector: 214 nm

Run Time: 45 min

Post Time: 15 min

Temperature: Ambient

B. Tissue Factor Chromogenic Assay

This assay determines the relative enzymatic activity in absorbanceunits of recombinant tissue factor per milliliter. The liquid tissuefactor and the liquid gel were diluted with assay diluent (50 mMTris.HCl, 100 mM NaCl, 0.1% BSA, and 0.01% thimerosal) and then withrelipidation buffer (800 μL thimerosal, 25 μl 1 100 mM CdCl₂, and 50 μlof a mixture of 0.5 g phosphatidylcholine and q.s. to 100 ml ofdeoxycholate) to 4 ng/ml, 2 ng/ml, and 0.5 ng/ml. Duplicate samples werethen submitted for analysis. The chromogenic tissue factor assayemployed is described in col. 10 of U.S. Pat. No. 5,017,556 issued May21, 1991.

C. Study No. 4: Effects of PEG and Glycerol on Gel Containing TissueFactor

Tissue factor was obtained as described in EP 278,776 published 17 Aug.1988 and formulated in1.1% octylglucoside in a buffer at pH 7.2. In thisstudy both the protein and the buffer were concentrated aboutthree-fold. The protein in hydroxyethyl cellulose gel was initiallysubmitted for the tissue factor chromogenic assay described above todetermine the feasibility of such a study. With promising results fromthis assay a size-exclusion HPLC stability study was set up. The sameexcipients and amounts thereof as were used in the relaxin Study No. 3were employed, as well as 50 mM PEG 3350. The two grades of PEG (300 and3350) exist as a liquid and as a solid, respectively, at roomtemperature. The protein concentration used in this study was 1 mg/ml. Athree-week stability study of the reconstituted gel was set up. The gelswere lyophilized with primary drying at -40° C. for 55 hours.

D. Results

This study demonstrates that glycerol protected the tissue factorprotein from degradation in a lyophilized gel formulation during a 5° C.storage. The two grades of PEG, on the other hand, did not show anyprotective properties for this protein. See FIG. 10.

EXAMPLE 3

This example illustrates the invention utilizing TGF-β as the protein.

A. Reverse-Phase HPLC Assay for TGF-β

Instrument: HP 1090

Column: Vydac C4

Mobile Phase: A) 0.1% TFA in water, B) 0.1% TFA in acetonitrile

Gradient: 27% to 35% B in 35 minutes

Flow Rate: 0.5 ml/min

Injection Volume: 200 μl

UV Detector: 214 nm

Run Time: 60 minutes

Post Time: 15 minutes

Temperature: 40° C.

B Study No. 5: Effects of Sucrose on Gel Containing TGF-β

Liquid TGF-β was added to premixed hydroxyethyl cellulose gel asdescribed above to reach a final protein concentration of 0.5 mg/ml,hydroxyethyl cellulose concentration of 3.5% w/v, and sucroseconcentration of 0.2M. The gel was then lyophilized and the resultingcakes were stored at 5° C. After a period of storage the lyophilized gelsample was reconstituted with water and assayed with the reverse-phaseHPLC assay described above.

C. Results

After eight days of storage at 5° C. the lyophilized gel product stillmaintained about 90% peak area of the liquid bulk standard, suggestingthat sucrose is a likely candidate excipient for stabilizing TGF-β inthe lyophilized hydroxyethyl cellulose gel.

Conclusions A. Reconstitution

Of the ten excipients examined, sucrose, sodium glutamate, and magnesiumsulfate reconstitute the lyophilized gel in under 10 minutes while theothers may take up to 2 hours. For sodium glutamate and PEG, the higherthe concentration the shorter the reconstitution time. On the otherhand, mannitol had the opposite effect. Also, the higher the molecularweight of PEG, the shorter its reconstitution time. As for glycerol,even though gels at the higher concentration have a reasonablereconstitution time, cake appearance is not acceptable.

B. Protein Stability

The results from the various studies conducted show that lyophilized gelis a stable environment for storing proteins if an appropriate excipientis selected. However, the excipient is protein specific. For example,glycerol keeps tissue factor stable in the lyophilized Natrosol gel, butsuch results were not observed with relaxin. Sucrose, on the other hand,stabilizes relaxin in a similar environment.

Summary

Based on the results from these reconstitution and protein stabilitystudies, it is apparent that proteins can be formulated with cellulosederivatives and lyophilized so as to obtain gels suitable for topicalapplications. As to excipients for the proteins, sucrose is a preferredcandidate for relaxin and TGF-β, whereas glycerol is a preferredcandidate for tissue factor, based on their ability both to preserveprotein stability and allow rapid reconstitution of the lyophilizedmixture to form a gel.

What is claimed is:
 1. A formulation comprising tissue factor, glycerol,and a water-soluble etherified cellulose in an amount that uponreconstitution of the mixture will form a topical gel.
 2. Theformulation of claim 1 wherein the glycerol concentration in thereconstituted gel is about 1-4% weight/volume.
 3. The formulation ofclaim 2 wherein the tissue factor concentration in the reconstituted gelis about 0.2 to 1 mg/ml.
 4. The formulation of claim 1 wherein theetherified cellulose is hydroxyethylcellulose or methylcellulose.
 5. Theformulation of claim 1 that is reconstituted to a gel with sufficientwater.
 6. The formulation of claim 5 wherein the etherified cellulose ispresent in an amount of about 1-10% by weight/volume of the gel.
 7. Theformulation of claim 6 wherein the etherified cellulose ishydroxyethylcellulose in an amount of about 1-5% by weight/volume of thegel.
 8. The formulation of claim 7 wherein the hydroxyethylcellulose ispresent in an amount of about 3-4% by weight/volume of the gel.
 9. Amulti-unit package comprising, in one unit, the formulation of claim 1,and in another unit, a diluent for the formulation.
 10. Adual-compartment syringe wherein one compartment contains theformulation of claim 1 and the other compartment contains a diluent forthe formulation.
 11. A method for treating tissue comprising the stepsof:(a) providing a formulation comprising a lyophilized mixture oftissue, factor, glycerol, and a water-soluble etherified cellulose in anamount that upon reconstitution of the mixture will form a topical gel,(b) reconstituting the mixture in a sufficient amount of water to form agel, and (c) applying a therapeutically effective amount of the geltopically to the tissue.
 12. The method of claim 11 wherein the tissueis skin.
 13. A method of modulating the reproductive physiology ofmammals during pregnancy and parturition comprising administering to thecervix or vagina a therapeutically effective amount of a gel that hasbeen reconstituted from a formulation comprising a lyophilized mixtureof relaxin, sucrose, and a water-soluble etherified cellulose in anamount that upon reconstitution of the mixture will form a topical gel.